ABSTRACT This is a renewal of ongoing R01 funding to define molecular sites of alcohol action in brain and to link alcohol effects on these receptors with specific behaviors such as consumption, reward/aversion, intoxication and physical dependence. During the current period of funding we were successful in providing molecular and even atomic level of analysis of alcohol sites on pentameric ligand-gated (pLGIC) ion channels, including the first crystallographic structure of alcohols bound within a channel protein. We also succeeded in constructing mutant mice with GABAa receptor subunits which are resistant to alcohol modulation allowing us to link specific GABAa receptors with discrete behavioral actions of alcohol. We are in the middle of the fourth year of this project period and to date have 30 publications with 7 more submitted or in preparation. Although we and others have made considerable progress, the molecular sites of alcohol action in the brain are not completely defined. Emerging evidence from human and rodent genetics, as well as recombinant receptors, indicates that several targets which have received only limited attention may be important sites of alcohol action in brain. We propose to define the role of two groups of ligand-gated ion channels, the glycine-activated chloride channels (GlyR) and GABAaRs formed from subunits ( GABAaRs) in actions of alcohol at the molecular, electrophysiological and behavioral levels. A strength of this proposal is the combined use of knock-out (KO) and knock-in (KI) mice allowing us to define the importance of the presence of a subunit (KO) and the importance of direct alcohol action on the protein (KI) for behavioral actions of alcohol. An innovative aspect is use of a new technology (transcription activator- like effector nuclease; TALEN) to be employed by our collaborator, Dr. Gregg Homanics, that markedly reduces the time and cost required for construction of mutant mice. In addition to our behavioral studies, mutant mice will be used by our collaborator, Dr. Neil Harrison for electrophysiological studies of glycinergic function in accumbal regions. Several GlyRs and GABAaRs are genetically linked with human alcohol dependence and understanding their role in alcohol actions may provide new targets for pharmacotherapies of alcohol abuse and alcoholism.